Nucleic acid molecules containing spacers and methods of use thereof

A method for producing a plurality of adeno-associated virus (AAV) particles, the method comprising:

• (a) transfecting a host cell with a vector, wherein said vector comprises a nucleic acid molecule comprising:
• (i) a first spacer (SS1);
• (ii) a first adeno-associated virus (AAV) inverted terminal repeat (ITR1);
• (iii) a heterologous polynucleotide molecule (HPM);
• (iv) a second AAV inverted terminal repeat (ITR2);
• and
• (v) a second spacer (SS2),
• wherein the components are operably linked to each other in a 5′-to-3′ direction as:
• SS1-ITR1-HPM-ITR2-SS2, wherein SS1 is directly adjacent to, and outside of, the ITR1 and the SS2 is directly adjacent to, and outside of, the ITR2, and wherein the host cell expresses or is modified to express an AAV capsid (Cap) protein and an AAV Rep protein;
• (b) culturing the host cell under conditions sufficient to allow for:
• (i) replication of the nucleic acid molecule, from ITR1 through ITR2, by the AAV Rep protein;
• and
• (ii) packaging of the replicated nucleic acid molecules resulting from (b) (i) by the AAV Cap protein, thereby producing the plurality of AAV particles;
• and
• (c) isolating the plurality of AAV particles from the host cell.